Grapevine fanleaf virus (GFLV) is considered one of the most prevalent and serious diseases infecting grapevine in the world. Visual observations were conducted in the field concerning the GFLV symptoms in the elder and young trees in Beheira Governorate. Plants with characteristic symptoms of GFLV were collected during the growing season and checked by DAS- ELISA. Leaf samples that reacted positively to GFLV antiserum were used first to inoculate the test plants and then for biological purification of the virus isolate using single local lesions developed on Chenopodium quinoa, whereas, Gomphrena globosa was used as a source for virus inoculum. Biological indexing was carried out using woody indicator cutting of virus-free rootstocks; freedom and LN33. RT-PCR was done to confirm the ELISA results. Anatomical changes in the infected cells of leaves compared with healthy ones were studied. The infection rate of the virus was 10.29%. There are quantifiable changes in physiological and biochemical markers such as proteins, pigment and carbohydrate content, phenolic compounds, polyphenol oxidase (PPO), peroxidase (POX), and catalase (CAT) activity in grapevine leaves. Infected grapevine leaves had higher levels of phenolic compounds, PPO, POX, and CAT than healthy leaves, although total protein content and pigment levels were in the other direction. Overall, the findings imply that GFLV infection causes considerable changes in enzyme levels that result in the development of symptoms that cannot be reversed. The thermotherapy at 36^oC for 60 days in combination with meristem tip cultures resulted in 100% virus-free cultures.